The invention relates to methods and devices for treating various medical conditions, e.g., lung dysfunction. Some of the conditions are related to cystic fibrosis, and others may be more widely applicable.
Cystic fibrosis (CF) is the most common life-shortening genetic disease in the white population. It occurs in the USA in about {fraction (1/3,300)} white births, {fraction (1/15,300)} black births, and {fraction (1/32,000)} Asian-American births; 30% of patients are adults. See, e.g., Berkow (ed.) The Merck Manual of Diagnosis and Therapy Merck and Co., Rahway, N. J.; Thorn, et al. Harrison""s Principles of Internal Medicine McGraw-Hill, N.Y.; and Weatherall, et al. (eds.) Oxford Textbook of Medicine Oxford University Press, Oxford; the Cystic Fibrosis Foundation website (www.cff.org); Davis (ed. 1993) Cystic Fibrosis Marcel Dekker, ISBN: 082478815X; Dodge (ed. 1996) Cystic Fibrosis: Current Topics Wiley and Son, ISBN: 0471963534; Bauernfeind, et al. (eds. 1996) Cystic Fibrosis Pulmonary Infections: Lessons from Around the World (Respiratory Pharmacology and Pharmacotherapy) Springer Verlag, ISBN: 081765027X; Orenstein and Stern (eds. 1998) Treatment of the Hospitalized Cystic Fibrosis Patient Marcel Dekker, ISBN: 0824795008; Hodson, et al. (eds. 2000) Cystic Fibrosis Oxford Univ. Press, ISBN: 0340742089; and Yankaskas and Knowles (eds. 1999) Cystic Fibrosis in Adults Lippincott Pubs, ISBN: 0781710111. See also Conese and Assael (2001) xe2x80x9cBacterial infections and inflammation in the lungs of cystic fibrosis patientsxe2x80x9d Ped. Infect. Dis. J. 20:207-213; Moss (2001) xe2x80x9cNew approaches to cystic fibrosisxe2x80x9d Hosp. Pract. (Off. Ed.) 36:25-27, 31-32, 35-37; Robinson (2001) xe2x80x9cCystic fibrosisxe2x80x9d Thorax 56:237-241; Ratjen (2001) xe2x80x9cChanges in strategies for optimal antibacterial therapy in cystic fibrosisxe2x80x9d Int. J. Antimicrob. Agents 17:93-96; Hodson (2000) xe2x80x9cTreatment of cystic fibrosis in the adultxe2x80x9d Respiration 67:595-607; Doring, et al. (2000) xe2x80x9cAntibiotic therapy against Pseudomonas aeruginosa in cystic fibrosis: a European consensusxe2x80x9d Eur. Respir. J. 16:749-767; Beringer and Appleman (2000) xe2x80x9cUnusual respiratory bacterial flora in cystic fibrosis: microbiologic and clinical featuresxe2x80x9d Curr. Opin. Pulm. Med. 6:545-550; Larson and Cohen (2000) xe2x80x9cCystic fibrosis revisitedxe2x80x9d Mol. Genet. Metab. 71:470-477; Nasr (2000) xe2x80x9cCystic fibrosis in adolescents and young adultsxe2x80x9d Adolesc. Med. 11:589-603; Rahman and MacNee (2000) xe2x80x9cOxidative stress and regulation of glutathione in lung inflammationxe2x80x9d Eur. Respir. J. 16:534-554; and Koch and Hoiby (2000) xe2x80x9cDiagnosis and treatment of cystic fibrosisxe2x80x9d Respiration 67:239-247.
CF is typically carried as an autosomal recessive trait by about 3% of the white population. The most common gene responsible has been localized to 250,000 base pairs of genomic DNA on chromosome 7q (the long arm). This encodes a membrane-associated protein called the cystic fibrosis transmembrane regulator (CFTR). The most common gene mutation, F508, leads to absence of a phenylalanine residue at position 508 on the CFTR protein and is found on about 70% of CF alleles;  greater than 600 less common mutations account for the remaining 30%. Although the exact function of CFTR is unknown, it appears to be part of a cAMP-regulated Clxe2x88x92 channel and appears to regulate Clxe2x88x92 and Na+ transport across epithelial membranes. Heterozygotes may show subtle abnormalities of epithelial transport but are clinically unaffected.
Fifty percent of patients present with pulmonary manifestations, usually chronic cough and wheezing associated with recurrent or chronic pulmonary infections. Cough is the most troublesome complaint, often accompanied by sputum, gagging, vomiting, and disturbed sleep. Intercostal retractions, use of accessory muscles of respiration, a barrel-chest deformity, digital clubbing, and cyanosis occur with disease progression. Upper respiratory tract involvement includes nasal polyposis and chronic or recurrent sinusitis. Adolescents may have retarded growth, delayed onset of puberty, and a declining tolerance for exercise. Pulmonary complications in adolescents and adults include pneumothorax, hemoptysis, and right heart failure secondary to pulmonary hypertension.
Cystic fibrosis still presents major health problems to afflicted individuals. It is a highly debilitating condition, and affects significant numbers of patients. As such, there is great need for new and more effective treatments. The present invention addresses these and other problems.
The present invention provides methods method of treating a respiratory tract infection in a primate suffering from cystic fibrosis or other respiratory tract condition, comprising administering to the primate an effective amount of aerosolized thiocyanate or halides. In certain embodiments, the treating is after symptoms of bacterial infection have been detected or perhaps for viral infections instead or for bacterial infections that follow viral infections of the respiratory tract. Typically, the lung infection is: Staphylococus aureus; Pseudomonas aeruginosa; or Burkholeria cepacia. The method may be used in combination, e.g., with a peroxidase; H2O2; and/or another treatment for a lung infection or cystic fibrosis. Such other treatments may be, e.g., an antibiotic; an antiviral; an enzyme; or a manipulation.
In another embodiment, the invention provides methods of treating a respiratory infection, e.g., lung, in a mammal, comprising administering to the mammal an effective amount of thiocyanate or other halides, e.g., Ixe2x88x92, Brxe2x88x92, etc. Often, the administering is: by aerosol inhalation of the thiocyanate; or in combination with: an antibiotic; an antiviral; an enzyme; H2O2; or a medical manipulation. The lung infection is likely to be: Staphylococus aureus; Pseudomonas aeruginosa; or Burkholeria cepacia. In various embodiments, the effective amount of thiocyanate is between about 5 xcexcM and 4 mM in the lung fluid; or the administering occurs between one administration in a week to hourly.
Another embodiment includes methods of treating a lung condition in a primate or rodent suffering from symptoms of cystic fibrosis, the method comprising administering to the lung of the creature an effective amount of thiocyanate. Typically, the administering is by inhalation of aerosolized thiocyanate; the creature is a mouse or rat; the creature suffers from a lung infection; or the treating further includes administration of a peroxidase or H2O2.
Yet another embodiment encompasses an inhaler comprising: thiocyanate, or a peroxidase and labeled, e.g., instructions, for administration to an individual with cystic fibrosis. The inhaler may further comprise an antibiotic or antiviral therapeutic. The individual may have a lung infection.
Outline
I. Cystic Fibrosis
II. Current CF Treatments
III. The Lactoperoxidase (LPO) System
IV. Therapeutic Applications
In accordance with the objects outlined above, the present invention provides novel methods for treating or preventing signs or symptoms of respiratory dysfunction, particularly infections resulting from compromised airway functions related to resistance to infectious agents which enter the body through the airway mucosa. Devices are provided which are useful in addressing these problems.
Descriptions of lung immunity is applicable to other parts of the respiratory tract. Since the mechanisms described herein are common to airways in genera, the applications described would also apply to other airway mucosal surfaces.
I. Cystic Fibrosis
Cystic fibrosis is the most common genetic disorder among Caucasians. The disease is characterized by chronic respiratory infection that begins early in life with Staphylococcus aureus and Haemophilus influenzae infections and later colonization with mucoid strains of Pseudomonas aeruginosa. Chronic respiratory infection results in progressive loss of lung function and greatly diminished quality of life or shortened life expectancy. See references listed in the Background section. Chronic infection implies failure of airway host defense against bacterial colonization although the exact defects in host defense remain unclear. The normal airway barrier to infection is complex and multifactorial. The currently accepted view is that cilia mechanically clear potential infectious particles and that mucus provides both a protective barrier and serves as a carrier with the proper viscoelastic properties to permit ciliary movement. The protective barrier of mucus is thought to be both physical and chemical as a number of anti-microbial components are secreted by airway epithelial and submucosal gland cells. Lung physiology and function are fundamental clinical study areas, and are described, e.g., in Murray and Nadel (2001) Textbook of Respiratory Medicine Saunders, Philadelphia, Pa. (ISBN: 0721692532); Crystal, et al. (1996) The Lung Lippincott Williams and Wilkins, Philadelphia, Pa. (ISBN 0-397-51632-0); Brewis, et al. (1995) Respiratory Medicine 2d Ed., Saunders, Philadelphia, Pa. (ISBN: 072016411); and Fraser, et al. (1988) Diagnosis of Diseases of the Chest Volume 3d Ed., Saunders, Philadelphia, Pa. (ISBN: 0721638708).
Additionally, nearly all exocrine glands are affected in varying distribution and degree of severity. Involved glands are of 3 main types: those that become obstructed by viscid or solid eosinophilic material in the lumen (e.g., pancreas, intestinal glands, intrahepatic bile ducts, gallbladder, submaxillary glands); those that are histologically abnormal and produce an excess of secretions (e.g., tracheobronchial and Brunner""s glands); and those that are histologically normal but secrete excessive Na+ and Clxe2x88x92 (e.g., sweat, parotid, and small salivary glands). Duodenal secretions are viscid and contain an abnormal mucopolysaccharide. The reproductive functions for both adult males and females are typically also affected.
Morbidity in cystic fibrosis (CF) primarily results from chronic respiratory infections by Pseudomonas aeruginosa. Mutations in CFTR result in an anion channel defect but how this leads to impaired host defense against infection is not fully understood. Evidence suggests that the lungs are histologically normal at birth. Pulmonary damage is probably initiated by diffuse obstruction in the small airways by abnormally thick mucus secretions. Bronchiolitis and mucopurulent plugging of the airways occur secondary to obstruction and infection. Bronchial changes are more common than parenchymal changes. Emphysema is not prominent. As the pulmonary process progresses, bronchial walls thicken; the airways fill with purulent, viscid secretions; areas of atelectasis develop; and hilar lymph nodes enlarge. Chronic hypoxemia results in muscular hypertrophy of the pulmonary arteries, pulmonary hypertension, and right ventricular hypertrophy. Much of the pulmonary damage may be caused by immune-mediated inflammation secondary to the release of proteases by neutrophils in the airways. Bronchoalveolar lavage fluid, even early in life, contains large numbers of neutrophils and increased concentrations of free neutrophil elastase, DNA, and interleukin-8.
Early in the course, Staphylococcus aureus is the pathogen most often isolated from the respiratory tract, but as the disease progresses, Pseudomonas aeruginosa is most frequently isolated. A mucoid variant of Pseudomonas is uniquely associated with CF. Colonization with Burkholderia cepacia occurs in up to 7% of adult patients and may be associated with rapid pulmonary deterioration.
II. Current CF Treatments
Many of the signs and symptoms of CF are pulmonary. Thus, chest x-ray findings often aid diagnosis. Hyperinflation and bronchial wall thickening are typically the earliest findings. Subsequent changes include areas of infiltrate, atelectasis, and hilar adenopathy. With advanced disease, segmental or lobar atelectasis, cyst formation, bronchiectasis, and pulmonary artery and right ventricular enlargement occur. Branching, fingerlike opacifications that represent mucoid impaction of dilated bronchi are characteristic. In almost all cases, sinus x-rays and CT studies show persistent opacification of the paranasal sinuses.
Pulmonary function tests typically reveal hypoxemia and reduction in forced vital capacity (FVC), forced expiratory volume in 1 sec (FEV1), and FEV1/FVC ratio and an increase in residual volume and the ratio of residual volume to total lung capacity. Fifty percent of patients have evidence of airway hyperreactivity.
Current treatment for pulmonary manifestations includes prevention of airway obstruction and prophylaxis against and control of pulmonary infection. Prophylaxis against pulmonary infections consists of maintenance of pertussis, and immunity to Haemophilus influenzae, varicella, measles, and influenza by vaccination. In unvaccinated patients, amantadine can be used for prophylaxis against influenza A.
Chest physical therapy consisting of postural drainage, percussion, vibration, and assisted coughing is recommended at the first indication of pulmonary involvement. In older patients, alternative airway clearance techniques such as active cycle of breathing, autogenic drainage, flutter valve device, positive expiratory pressure mask, and mechanical vest therapy may be effective. For reversible airway obstruction, bronchodilators may be given orally, and/or by aerosol and corticosteroids by aerosol. O2 therapy is indicated for patients with severe pulmonary insufficiency and hypoxemia. Noninvasive positive pressure ventilation by nasal or facemask also can be beneficial. Long-term daily aerosol administration of dornase alfa (recombinant human deoxyribonuclease) has been shown to slow the rate of decline in pulmonary function and to decrease the frequency of severe respiratory tract exacerbations. Other aspects of current treatment are known. See, e.g., The Merck Manual, and CF references listed above.
Drug therapies include oral corticosteroids for infants with prolonged bronchiolitis and in those patients with refractory bronchospasm, allergic bronchopulmonary aspergillosis, and inflammatory complications (e.g., arthritis and vasculitis). Long-term use of alternate-day corticosteroid therapy can slow the decline in pulmonary function, but because of steroid-related complications it is not recommended for routine use. Patients receiving corticosteroids must be closely monitored for evidence of carbohydrate abnormalities and linear growth retardation. Ibuprofen, when given at a dose sufficient to achieve a peak plasma concentration between 50 and 100 xcexcg/ml over several years, has been shown to slow the rate of decline in pulmonary function, especially in children 5 to 13 yr. The appropriate dose must be individualized based on pharmacokinetic studies.
Antibiotics should be used in symptomatic patients to treat bacterial pathogens in the respiratory tract, according to culture and sensitivity testing. A penicillinase-resistant penicillin (e.g., cloxacillin or dicloxacillin) or a cephalosporin (e.g., cephalexin) is the drug of choice for staphylococci. Erythromycin, amoxicillin-clavulanate, ampicillin, tetracycline, trimethoprim-sulfamethoxazole, or occasionally chloramphenicol may be used individually or in combination for protracted ambulatory therapy of pulmonary infection due to a variety of organisms. Ciprofloxacin is effective against sensitive strains of Pseudomonas. For severe pulmonary exacerbations, especially in patients colonized with Pseudomonas, parenteral antibiotic therapy is advised, often requiring hospital admission but safely conducted at home in carefully selected patients. Combinations of an aminoglycoside (tobramycin, gentamicin) with an anti-Pseudomonas penicillin are given IV. Intravenous administration of cephalosporins and monobactams with anti-Pseudomonas activity also may be useful. Serum aminoglycoside concentrations should be monitored and dosage adjusted to achieve a peak level of 8 to 10 xcexcg/ml (11 to 17 xcexcmol/L) and a trough value of  less than 2 xcexcg/ml ( less than 4 xcexcmol/L). The usual starting dose of tobramycin or gentamicin is 7.5 to 10 mg/kg/day in 3 divided doses, but high doses (10 to 12 mg/kg/day) may be required to achieve acceptable serum concentrations. Because of enhanced renal clearance, large doses of some penicillins may be required to achieve adequate serum levels. The goal of treating pulmonary infections should be to improve the clinical status sufficiently so that continuous use of antibiotics is unnecessary. However, in some ambulatory patients with frequent pulmonary exacerbations, long-term use of antibiotics may be indicated. In selected patients, long-term tobramycin therapy by aerosol may be effective.
Aerosol therapy with ribavirin should be considered in infants with respiratory syncytial viral infection. Levels of airway glutathione have been shown to be reduced in the airway lavages and in plasma of cystic fibrosis patients. In vitro studies of airway lining cells from normal and cystic fibrosis patients have shown that cystic fibrosis cells are defective in glutathione transport compared to normal and that replacement of the defective gene product restores glutathione transport. Since the LPO system can oxidize glutathione and since microorganisms have specific uptake systems for glutathione, and since glutathione is deficient in cystic fibrosis, combination therapies with all or part of the LPO system and glutathione may provide increased efficacy or may be necessary for system function.
That lung infections are problematic in CF is evidenced by the recent activity in this area. The first new drug therapy developed exclusively for CF in 30 years was approved by the Food and Drug Administration (FDA) in 1993. In clinical trials, this mucus-thinning drug called Pulmozyme(copyright), reduced the number of respiratory infections and improved lung function. In 1995, a four-year CF Foundation-supported study showed that the drug, ibuprofen, reduced the rate of lung inflammation in children with CFxe2x80x94under controlled conditions, and in high doses. This is a DNAse that liquifies secretions that are containing large amounts of bacterial DNA.
In late 1997, the FDA approved the drug TOBI(trademark) (tobramycin solution for inhalation). In clinical trials, this reformulated version of the common antibiotic improved lung function in people with CF and reduced the number of hospital stays. The benefits of TOBI(trademark) are that it can be delivered in a more concentrated dose directly to the site of CF lung infections more efficiently, and that it is preservative-free. The development of TOBI(trademark) should lead to a long line of other aerosolized antibiotics for people with CF.
Beyond currently available antibiotics, the CF Foundation pursues novel strategies that will lead to entirely new forms of antibiotics, i.e., to clear lung infections. The promising compound, IB367, represents one of an up-and-coming new class of drugs that should provide physicians unique tools to better manage chronic CF lung infections.
In CF cells, salt does not move properly because the protein product of the CF gene is defectivexe2x80x94and makes a faulty channel for the salt (chloride) to exit. Scientists are therefore looking for ways to get the chloride out of cells. INS365 is being evaluated for its ability to stimulate cells to secrete chloride. This, in turn, should lead to mucus that is less thick and sticky.
Other therapeutics in development are described in the Cystic Fibrosis Foundation website. Among Phase III clinical trial candidates are protein-assist and chloride channel strategy therapeutics and anti-inflammatory and anti-infection therapeutics.
The protein-assist and chloride channel strategies attempt to correct the defective CFTR protein or at least get enough protein to the cell surface, where it can operate as a channel. For people who do not have CF, the CFTR protein acts as a xe2x80x9cone-way doorxe2x80x9d that allows cells to release chloride. However, for people with CF, this protein is defective, which leads to improper chloride balance and the thick, sticky CF mucus. Chloride channel therapies are another exciting method of correcting the defect in CF cells. Researchers believe that getting CF cells to release chloride would be of a tremendous therapeutic benefit for CF patients.
Anti-inflammatory and anti-infection therapies are directed to medications that help control infection and inflammation because these CF symptoms tear down airways in a dangerous cycle. When CF lungs become infected, they also become over-inflamed. This over-inflammation leads to a greater susceptibility to future infection. Breaking or preventing this destructive cycle is a key focus for CF research. Although these therapies do not target the basic defect in CF cells, there are many exciting medications being evaluated that could extend lives by stemming the course of dangerous infections and the resulting accumulation of damage.
The present invention provides another therapeutic. While not limited according to mechanism, the present invention would seem to be based, in part, upon insight into the natural systems of lung immunity which appear to be compromised in CF.
III. The Lactoperoxidase (LPO) System
The lactoperoxidase (LPO) antibiotic system, shown to participate in ovine airway host defense, requires the anion thiocyanate (SCNxe2x88x92) in airway secretions for its function. To test the hypothesis that CF mutations lower airway [SCNxe2x88x92] thereby leading to impaired LPO-mediated host defense, human airways were examined for an LPO system. Normal and CF epithelial cell cultures were compared for differences in SCNxe2x88x92 transport that could be functionally linked to CF. LPO and SCNxe2x88x92 were found in human airway secretions and LPO mRNA was found in trachea tissue. In air liquid interface cultures, normal human airway epithelia transported SCNxe2x88x92 from the basolateral to the apical compartment and concentrated the anion at the mucosal surface with pharmacological properties consistent with CFTR-mediated channel activity. The pharmacological characteristics of SCNxe2x88x92 transport implied at least an indirect role of CFTR. SCNxe2x88x92 transport and apical SCNxe2x88x92 accumulation were significantly reduced in CF cultures. This alteration in SCNxe2x88x92 transport suggests a simple mechanistic link between CF mutations and the LPO host defense system in human airway.
The cystic fibrosis gene product, cystic fibrosis transmembrane regulator (CFTR), contains an anion channel that has been extensively characterized and mutations result in loss of functional CFTR anion channel activity on the apical cell surface. The current controversy regarding the link between cystic fibrosis and defective airway host defense has been reviewed by others and centers on the exact determination of the ionic composition of airway surface liquid (ASL) and the volume of ASL in cystic fibrosis versus normal airways. opposing views focus on either the effects of dehydration and impaired ciliary clearance of mucus or the effects of hypertonic ASL on the activity of antimicrobials in mucus. Both views are limited by our incomplete knowledge regarding the total array of mechanisms at work in airway host defense.
For example, the lactoperoxidase (LPO) system has recently been shown to be important to clear bacteria from sheep airways (Gerson, et al. (2000) Am. J. Respir. Cell Mol. Biol. 22:665-671) although its existence in human airways has not previously been shown. The LPO antibiotic system works to preserve milk sterility and contributes antibiotic activity to saliva. LPO uses H2O2 to catalyze oxidation of the anions SCNxe2x88x92 (a psuedohalide) and some halides (e.g., Ixe2x88x92 and Brxe2x88x92) to the antibiotic forms (e.g., OSCNxe2x88x92, OIxe2x88x92, or OBrxe2x88x92). Thomas, et al. (1991), pp. 123-142, in Everse, et al (eds.) Peroxidases in Chemistry and Biology. Descriptions will be directed to SCNxe2x88x92, but similarly, Ixe2x88x92 and Brxe2x88x92 may be substituted. 
Since it is known that the LPO system is effective against pseudomonads (Bjorck, et al. (1975) Applied Microbiology 30:199-204), and that SCNxe2x88x92 is a requisite substrate for LPO and is permeant through CFTR (Tabcharani, et al. (1993) Nature 366:79-82), defects in SCNxe2x88x92 transport could be, at least partially, responsible for the chronic respiratory bacterial infections seen in CF patients. Described experiments herein support the hypothesis that the LPO system is present in human airways and that cystic fibrosis airway epithelia are defective in SCNxe2x88x92 transport to the airway surface.
The experiments presented here document the presence of the lactoperoxidase anti-microbial system in human airways that was not previously recognized as a factor in human airway host defense. The data also show that the secretion of LPO""s substrate, SCNxe2x88x92, appears to rely, at least indirectly, on the CFTR anion channel and that mutant CFTR results in defective delivery of LPO""s substrate to the airway lumen.
Pseudomonas infection occurs with high frequency in CF airways and the LPO system""s efficacy against pseudomonads has been previously documented. Bjorck, et al. (1975) Applied Microbiology 30:199-204. Defective SCNxe2x88x92 transport in CF may impair the correct functioning of the LPO antibiotic system and be a contributor to the characteristic chronic respiratory infections seen in this disease. SCNxe2x88x92 is elevated in CF sweat (Gibbs and Hutchings (1961) Proc. Soc. Exp. Biol. Med. 106,:368-369) and others have suggested that SCNxe2x88x92 might be a pathogenic factor in CF based on decreased serum levels in severely affected CF patients compared to normal individuals (which would predict an even lower concentration in CF airways than that calculated above). Weuffen, et al. (1991) Padiatrie und Grenzgebiete 30:205-210.
Several mouse models of cystic fibrosis have been developed and none show the characteristic chronic airway infection seen in the human disease. Murine respiratory tracts were examined for the presence of LPO or an LPO-like protein by collecting tracheal secretions and assaying enzyme activity. Attempts to demonstrate an LPO system in airways of normal mice failed, e.g., in mice whose airways were challenged with live bacteria, or in mice previously sensitized and challenged with ovalbumin in order to up-regulate secretory cells in the airways. Since LPO is made by airway submucosal glands and goblet cells (Gerson, et al. (2000) Am. J. Respir. Cell Mol. Biol. 22:665-671; and Salathe, et al. (1997) Am. J. Resp. Cell and Mol. Biol. 17:97-105), the lack of these structures in mouse airways suggested that LPO may not be present in this animal""s respiratory tract and that mice may rely on an alternate airway defense. Lack of reliance on an LPO system could explain, in part, why CFTR knockout mice do not display the chronic respiratory infection characteristic of cystic fibrosis.
One unexplained feature of chronic infection in CF is the inability of neutrophils to clear bacteria. CF airways are characterized by a constant high level of lumenal neutrophils that appear to be competent in phagocytosis, chemotaxis, and have functional myeloperoxidase (MPO). Yet bacterial infection is not resolved even in the presence of aggressive antibiotic therapies, suggesting a defect in neutrophil killing. Although mucoid strains of Pseudomonas are present in later stages of CF, defects in host defense and neutrophil mediated killing exist in earlier stages of the disease suggesting that mucoid character alone does not explain the inability of neutrophils to resolve infections. MPO uses Brxe2x88x92, Ixe2x88x92, Clxe2x88x92, and SCNxe2x88x92 as substrates and although most studies of MPO-mediated bacterial killing have concentrated on use of Clxe2x88x92, SCNxe2x88x92 has recently been shown to be the preferred substrate of MPO. Thus impaired SCNxe2x88x92 transport to the lumen could potentially result in defects in both the epithelial-derived LPO system as well as the neutrophil-derived MPO antibacterial activity. The SCNxe2x88x92 deficiency be doubly detrimental to maintenance of a sterile airway.
Because the LPO antimicrobial system relies on correct SCNxe2x88x92 anion concentration rather than osmolarity of ASL or water volume, measures of these ASL characteristics are not expected to be relevant to LPO antibacterial activity. The total loss of airway lumenal SCNxe2x88x92 would probably not be detected in measurement of ASL ionic strength or tonicity as it is xe2x89xa61 mM in ASL of normal airways. Elemental analysis of ASL has demonstrated large amounts of sulfur that have been ascribed to the presence of sulfated mucins and that may obscure the detection of SCNxe2x88x92 in these studies.
Taken together with the extensive literature on mutant CFTR-linked changes in the airway, the data presented here suggest that defective SCNxe2x88x92 transport to the airway may account for a portion of the chronic infection seen in cystic fibrosis and that aerosol SCNxe2x88x92 therapy may bolster airway host defense against infection in patients with this disease.
IV. Therapeutic Application
According to the present invention, methods for treating various respiratory system conditions are provided, particularly those related to respiratory system response to infectious agents. These conditions are typical of the normal or dysfunctional respiratory tract in a mammal, e.g., primate, sheep, rodent, or common domestic pet, including dogs and cats. The present disclosure provides evidence for a natural response to airway infections in primates. This system, the LPO system, is implicated as a mechanism to decrease likelihood of established colonization of microbes in the lung.
These reagents can be combined for therapeutic use with additional active or inert ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, along with physiologically innocuous stabilizers, excipients, or preservatives. These combinations can be sterile filtered and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations.
The LPO system is found in the lung of at least some species. In those species, including certain primates, the presence of that system should provide antibacterial function, which should be generally effective against most common infectious bacterial and fungal species. The enzymatic conversion of the anion SCNxe2x88x92 to the antimicrobial OSCN-results in a means to decrease microbial load to the lung. Other species, which lack the LPO system, may serve as species useful for testing whether the introduction of such a system may be of value in certain lung infection models.
Since the lung fluid, which bathes the mucosa, is the target of treatment, an aerosol is the preferred method of administration. While other means to administer to the lung will be available, there is a great deal of inhaler art. Here, with the administration of small molecules, or perhaps in combination with a biologic, the inhalers are preferred means for administration. See, e.g., U.S. Pat. No. 6,223,746 xe2x80x9cMetered dose inhaler pumpxe2x80x9d; U.S. Pat. No. 6,205,999 xe2x80x9cMethods and apparatus for storing chemical compounds in a portable inhalerxe2x80x9d; U.S. Pat. No. 6,204,054 xe2x80x9cTranscytosis vehicles and enhancers for drug deliveryxe2x80x9d; U.S. Pat. No. 6,202,643 xe2x80x9cCollapsible, disposable MDI spacer and methodxe2x80x9d; U.S. Pat. No. 6,196,219 xe2x80x9cLiquid droplet spray device for an inhaler suitable for respiratory therapiesxe2x80x9d; U.S. Pat. No. 6,196,218 xe2x80x9cPiezo inhalerxe2x80x9d; U.S. Pat. No. 6,182,655 xe2x80x9cInhaler for multiple dosed administration of a pharmacological dry powderxe2x80x9d; U.S. Pat. No. 6,180,663 xe2x80x9cTherapeutic nasal inhalantxe2x80x9d; U.S. Pat. No. 6,176,238 xe2x80x9cDispenser for substances in powder or granular formxe2x80x9d; U.S. Pat. No. 6,170,482 xe2x80x9cInhalation apparatusxe2x80x9d; U.S. Pat. No. 6,165,484 xe2x80x9cEDTA and other chelators with or without antifungal antimicrobial agents for the prevention and treatment of fungal infectionsxe2x80x9d; U.S. Pat. No. 6,164,275 xe2x80x9cInhaler carrierxe2x80x9d; U.S. Pat. No. 6,158,428 xe2x80x9cInfant inhalerxe2x80x9d; U.S. Pat. No. 6,155,251 xe2x80x9cBreath coordinated inhalerxe2x80x9d; U.S. Pat. No. 6,153,224 xe2x80x9cCarrier particles for use in dry powder inhalersxe2x80x9d; U.S. Pat. No. 6,153,173 xe2x80x9cPropellant mixture for aerosol formulationxe2x80x9d; U.S. Pat. No. 6,149,892 xe2x80x9cMetered dose inhaler for beclomethasone dipropionatexe2x80x9d; U.S. Pat. No. 6,142,146 xe2x80x9cInhalation devicexe2x80x9d; U.S. Pat. No. 6,142,145 xe2x80x9cInhalation devicexe2x80x9d; U.S. Pat. No. 6,140,323 xe2x80x9cDosing method of administering medicaments via inhalation administration or skin administrationxe2x80x9d; and U.S. Pat. No. 6,138,673 xe2x80x9cInhalation device and methodxe2x80x9d. Many others may be found by a simple search through a patent database.
As such, the treatment described herein is intended to decrease the microbial load by a significant and measurable amount. While the normal lung may possess sufficient LPO to convert inhaled thiocyanate to an activated antimicrobial product, certain lung conditions may deplete either the thiocyanate or LPO, whose supplementation may overcome dysfunction resulting therefrom. Thus, the treatment is intended to decrease the microbial flora by at least about 10%, preferably at least about 20%, more preferably by at least about 50%, and better by at least 2, 3, 5, or 7 fold. Other relevant endpoints would be sputum neutrophil counts, sputum volume production, pulmonary function testing, incidence of hospitalization among a population of patients, or chest X-ray evaluation, which would improve according to treatment. Such measures will be taken at appropriate time points, as clinically significant. Such may be at specified time points after microbial exposure or introduction, and may range from minutes, hours, days, or weeks after defined identifiable events.
Treatment dosages should be initially titrated in development to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, e.g., in Gilman, et al. (eds.) Goodman and Gilman""s: The Pharmacological Bases of Therapeutics, latest Ed., Pergamon Press; and Remington""s Pharmaceutical Sciences, latest ed., Mack Publishing Co., Easton, Pa. Methods for administration are discussed therein and below, e.g., for direct application to the lung. Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, e.g., in the Merck Index, Merck and Co., Rahway, N.J. Dosage ranges would ordinarily be expected to deliver amounts that result in mucosal surface liquid concentrations lower than 10 mM concentrations, typically less than about 4 mM concentrations, preferably about 400 xcexcM, preferably not less than about 4 xcexcM. Slow release formulations, or a slow release apparatus may be utilized for continuous or long term administration. See, e.g., Langer (1990) Science 249:1527-1533. Dose evaluation may depend, e.g., on patient weight, age, condition, and other relevant variables. Exemplary dose ranges should account for total ASL and estimated sputum volumes. It is expected that normal amounts would be about 40 xcexcM. A range of 100 fold lower or higher should be tested, as the balance of excess activity vs. tolerable damage from excess may depend upon patient condition. Estimating ASL of about 10 ml and sputum volume is estimated to another 10 ml, then a dose of about [400] xcexcg is delivered in the volume of deposited aerosol.
Therapeutic formulations may be administered in many conventional dosage formulations. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation. Formulations typically comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Formulations include those suitable for lung administration. The formulations may conveniently be presented in unit dosage form and may be prepared by many methods well known in the art of pharmacy. See, e.g., Gilman, et al. (eds. 1990) Goodman and Gilman""s: The Pharmacological Bases of Therapeutics 8th Ed., Pergamon Press; and Remington""s Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Pa.; Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, New York; Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Tablets Dekker, New York; and Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker, New York. The therapy of this invention may be combined with or used in association with other agents, e.g., other lactoperoxidase substrates or therapies.
As indicated above, various combinations of therapeutics may be used, e.g., with antibiotics, antifungal agents, antiviral agents, with various peroxidases, with sulfhydryl compounds, with H2O2, or other therapeutics or procedures used in the treatment of CF or other lung infections. Such procedures include airway clearance techniques (ACTs) and physiotherapy, e.g., breathing exercises, postural drainage, chest percussion, vibration, assisted coughing, and forced expiratory techniques. Other lung conditions will include those where infections may be problematic, e.g., tuberculosis, asthmatic conditions, artificially ventilated patients, HIV or other immunosuppressed individuals. As described above, there are various treatments for cystic fibrosis, which may be combined with the treatment described herein. Administration may be simultaneous or sequential, e.g., with the various other therapeutics, or with the DNAse (Pulmozyme(copyright)) or TOBI(trademark).
Certain precautions would be indicated when administering thiocyanate. To limit the possibility of thiocyanate toxicity, other sources of thiocyanate should carefully monitored. Dietary sources include consumption of cauliflower, brussel sprouts, and other cyanogenic compounds, e.g., various seeds from squashes such as pumpkin. Smoking, exposure to welding fumes, or diesel exhaust should be controlled. The monitoring of blood thiocyanate levels may be useful, including determination of swallowed and absorbed compound. Serum and urine thiocyanate should be measured at appropriate times, e.g., 1, 4, 8 h following initial administration, and perhaps during treatment. Clinical parameters may also be monitored, e.g., body temperature, volume or rate of respiration, various subjective measures of lung infection or cystic fibrosis symptoms.
Side effects of treatment may include expected fever from bacterial lysis. Excessive levels of SCNxe2x88x92 may itself lead to toxic effects on the host.
Indications for use of the invention will include other respiratory tract conditions, e.g., beyond CF. Because of the ease of administration and low cost of the materials in the treatment described, the supplementation of the endogenous barrier to lung infections may be useful in many contexts. The circumstances in which lung infections may be problematic are well known in the art. See, e.g., Crystal, et al. (1996) The Lung Lippincott, ISBN 0-397-51632-0; and other lung references.
Thus, the present invention may become adopted as standard means to treat generic respiratory tract infections as a first and immediate step.